Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by progressive joint destruction. Initial destruction of cartilage and bone is associated with the formation of a pannus, consisting of a hypertrophic synovial membrane containing hyperplastic synoviocytes and an infiltrate of inflammatory cells including T cells, B cells, CD68+ macrophages, mast cells, and endothelial cells. The causes of RA are not well understood. Genetic studies have linked expression of specific major histocompatibility complex class II antigens to the development of RA, suggesting the involvement of antigen-specific mechanisms in disease progression (Zanelli et al., Hum. Immunol. 61:1254-1261 (2000)).
CD4+ T cells are thought to play a key role in initiation and progression of disease. Although many putative self antigens have been proposed, none have been definitively associated with the initiation of disease. Antigen-activated T cells stimulate monocytes, macrophages, and synovial fibroblasts to secrete pro-inflammatory cytokines including interleukin-1 (IL-1), interleukin-6 (IL-6), and TNF-α. These cytokines stimulate synovial fibroblasts, osteoclasts, and chondrocytes to release matrix metalloproteinases (MMPs) that destroy surrounding tissue. Activated CD4+ T cells stimulate osteoclastogenesis that can also contribute to joint damage. The activated T cells also stimulate B cells present in the synovium via the CD40 pathway to differentiate into antibody secreting cells producing rheumatoid factor, which may also contribute to disease pathology.
Many of the cytokines found in rheumatoid synovium have been directly linked to disease pathology. For example, TNF-α promotes inflammation by inducing secretion of other inflammatory cytokines including IL-1, IL-6, IL-8, GM-CSF, as well as by upregulating adhesion molecule expression on endothelial cells and synovial fibroblasts. These two events promote increased migration of lymphocytes including neutrophils, monocytes, and T cells into the synovium. Neutrophils release elastase and proteases that degrade proteoglycan and contribute to joint destruction. Therapies targeting TNF-α include the use of soluble TNF-α receptor (Etanercept) and neutralizing antibodies specific for TNF-α (Infliximab), and result in a significant decrease in the number of swollen joints, as well as the numbers of T cells and plasma cells in the synovium of RA patients. Such therapies also result in a decrease in the expression of VCAM-1 and IL-1 in the synovium of treated patients (Bathon, et al., New Engl. J. Med. 343:1586-1593 (2000); Lipsky, et al., New Engl. J. Med. 343:1594-1602 (2000); Richard-Miceli, et al., Biodrugs 15:251-259 (2001)).
IL-1 has also been closely linked to the pathophysiology of RA. IL-1 induces synovial cell proliferation and activates MMP and prostaglandin production in vitro (Mizel et al., Proc. Natl. Acad. Sci. USA 78:2474-2477 (1981)). In several mouse models of arthritis, IL-1 is believed to play a dominant role in cartilage destruction, whereas TNF-α is primarily proinflammatory (Joosten et al., J. Immunol. 163:5049-5055 (1999)). Transgenic mice constitutively expressing human IL-1α in various organs develop a severe polyarthritic phenotype with a predominance of neutrophils and macrophages in the diseased joints (Niki et al., J. Clin. Invest. 107:1127-1135 (2001)). Synovitis developed within two weeks of birth, followed by pannus formation and cartilage destruction within 8 weeks after birth. Treatment of RA patients with a natural inhibitor of IL-1, recombinant human IL-1 receptor antagonist (IL-1Ra), significantly reduced clinical symptoms and the rate of progressive joint damage (Jiang et al., Arthritis Rheum. 43:1001-1009 (2000); Bresnihan et al., Biodrugs 15:87-97 (2001)).
A number of studies have sought to identify genes whose expression is associated with the development of RA. cDNA microarrays have been used to compare expression profiles between tissue samples derived from RA and inflammatory bowel disease patients. Such studies have found that prominently upregulated genes in RA samples include: IL-6; the MMPs stromelysin-1, collagenase-1, gelatinase A, and human matrix metallo-elastase; tissue inhibitors of metalloproteinases, including TIMP-1 and TIMP-3; the adhesion molecule VCAM-1; and chemokines including MCP-1, MIF, and RANTES (Heller et al. Proc. Natl. Acad. Sci. USA 94: 2150-2155 (1997)). Further, a cDNA library has been generated from monocytes obtained from a RA patient with active disease (Stuhlmuller et al., Arthritis Rheum. 43:775-790 (2000)). Genes found to be upregulated in these cells include IL-1α, IL-1β, IL-6, TNF-α, growth-related oncogene α, macrophage inflammatory protein 2, ferritin, α1-antitrypsin, lysozyme, transaldolase, Epstein-Barr virus-encoded RNA 1-associated protein, thrombospondin 1, angiotensin receptor II C-terminal homologue, and RNA polymerase II elongation factor.
In one study, a cDNA library was generated by subtracting cDNA derived from noninflammatory osteoarthritis (OA) synoviocytes from cDNA derived from cultured RA fibroblastoid synoviocytes (Seki et al., Arthritis Rheum. 41:1356-1364 (1998)). Genes found to be constitutively overexpressed in the rheumatoid synoviocyte line include: chemokine stromal cell-derived factor 1α; adhesion molecule VCAM-1; interferon-inducible 56-kD protein; 2′-5′-oligoadenylate synthetase; Mac-2 binding protein; extracellular matrix components biglycan, lumican, and IGFBP5; and semaphorin VI.
Studies have also been conducted using suppression subtractive hybridization to identify genes that are highly expressed in RA synovium relative to OA synovium (Justen et al., Mol. Cell. Biol. Res. Comm. 3:165-172 (2000)). Genes found to be specifically upregulated in RA synovium include: cytoskeletal γ-actin; the extracellular matrix components fibronectin and collagen IIIα1; superficial zone protein; elongation factor α1; granulin precursor; interferon-γ inducible lysosomal thiol reductase; the protease cathepsin B; phospholipase A2 group IIA; and annexin II.
Accordingly, there is a continuing need to identify genes whose expression is associated with the development and progression of RA. The identification of such genes permits the development of clones expressing such genes, thereby permitting the identification of compounds capable of modulating the activity of such genes and/or their expression products. Such compounds may have therapeutic utility in the diagnosis and/or treatment of RA and related disease states. The present invention is directed to meeting these and other needs.